Characterization of immunoreactive dynorphin in human phaeochromocytomas.

The prodynorphin-derived opioids, dynorphin (DYN) and alpha-neoendorphin (alpha NE) were studied in 24 human phaeochromocytomas and related tumours. Nineteen tumours, extracted in HCl (0.1 mol/l), contained concentrations of immunoreactive DYN (ir-DYN) ranging from less than 0.5 to 794 pmol/g wet weight. None of the extracts in HCl contained ir-alpha NE (all less than 2.4 pmol/g). Sephadex G-50 gel filtration chromatography of ir-DYN in HCl (0.1 mol/l) extracts of six tumours revealed three small peaks of ir-DYN of higher molecular size (approximately 12,000, 6000 and 3000 daltons), a minor peak of ir-DYN eluting just after DYN(1-17), and a broad major peak, consisting of at least three components, which was significantly retarded and eluted after the salt volume of the column. High-pressure liquid chromatography (HPLC) of these extracts revealed multiple peaks of ir-DYN, most of which did not coelute with any synthetic DYN peptides. On both gel filtration chromatography and HPLC, one of the minor peaks coeluted with DYN(1-32). None of the peaks of ir-DYN coeluted with DYN(1-17) which had been acetylated using acetic anhydride. Extracts of the same tumours in acetic acid (0.1 mol/l) yielded similar values for ir-DYN content, but parallelism in the assay was improved. Sephadex G-50 chromatography revealed a different pattern of ir-DYN with a major peak coeluting with DYN(1-17) and, in two tumours, a minor peak coeluting with DYN(1-8). Studies with HPLC revealed, however, that substantial degradation of synthetic DYN occurred during extraction in acetic acid (0.1 mol/l) in spite of the precautions taken. Phaeochromocytomas frequently contain ir-DYN in concentrations which may approach that of the mammalian pituitary. These tumours did not, however, contain ir-alpha NE and, with the possible exception of a small amount of DYN(1-32), the ir-DYN present did not correspond with any known sequences. Thus, whilst prodynorphin is expressed in phaeochromocytomas, it does not seem to be processed to the usual end-products, and post-translational modifications therefore seem likely. Enzymatic degradation of DYN may occur during extraction in acetic acid (0.1 mol/l), and this medium should, therefore, be avoided in studies of such labile peptides.